In luminescence microscopy, certain dyes such as phosphors or fluorophors are used for the specific marking of samples such as cell parts in the investigation of biological specimens. The sample is illuminated with illumination radiation representing an excitation radiation and the luminescence radiation excited in this way is picked up with detectors. For example, a beam splitter and block filter are provided for this in the microscope, which split the luminescence radiation from the excitation radiation and enable a separate observation. Thanks to this method, it is possible to represent individual, differently colored cell parts in the microscope. In multiple luminescence, several parts of a specimen are colored at the same time with different dyestuffs which bind specifically to different structures of the specimen. Moreover, one can survey samples which luminesce without adding dyes. Luminescence is being used here as a term covering phosphorescence and fluorescence.
Thus, it is known how to use marking molecules or a marking substance which can be activated by means of optical radiation. These marking molecules can only be excited in the activated state to emit certain luminescence radiation. Non-activated marking molecules even after exposed to excitation radiation emit no or at least no noticeable luminescence radiation. The activation radiation thus places the marking substance in a state in which it can be excited to luminesce. Other activation is also possible, such as thermal activation. The activation radiation is applied so that at least a certain fraction of the activated marking molecules are at a distance from neighboring activated molecules. After recording of the luminescence radiation, one then determines the center of their resolution-limited radiation distribution and from this determines mathematically the position of the molecules with high precision.
In luminescence microscopy, a position determination is performed with optical recording devices, such as highly sensitive cameras with a precision in the nanometer range. Various image evaluation methods are known for a localization of the marking molecules. But a high localization accuracy is only achieved laterally, i.e., in a plane coordinated with the image plane of the recording device. Thus, in this respect, the methods are limited to a two-dimensional analysis of the sample, and the plane is called, for example, the xy-plane.
For the localization of luminescent marking molecules in the third dimension, in the depth direction with respect to the imaging of the sample, designated as z for example, there are techniques known in the prior art. After the image recording of a layer, also known as a frame, the sample or an objective of the recording device is shifted in the depth direction in order to record another image of the next layer. The image recordings are then combined into a layer image comprising all the layers. In order to produce a layer image, each image recording must be tied in with the position of the sample in the depth direction.
A distance in the depth direction from one image recording to another should not be greater than a depth focus of the objective, also known as the capture region, for otherwise there will be gaps in the layer image. Therefore, a certain number of images have to be taken on account of the localization of the marking molecules in order to obtain a high-quality layer image.
An additional problem arises when making use of marking molecules. The excitation radiation or activation radiation is not confined to the image region of an individual image, that is, marking molecules are also excited or activated above and below the image region. These marking molecules are therefore not available for a further image recording in a higher or lower layer. The sample is bleached by the excitation radiation or activation radiation.
In order to reduce this effect, it has been proposed by Mlodzianoski et al., Optics Express 19, 15009 (2011) to shift the sample in the depth direction during a measurement process of many individual image recordings, while the sample is exposed to excitation radiation or activation radiation during the measurement. In this way, the bleaching of the sample is distributed over all layers of the overall measurement process.
Determining the position of the sample in the depth direction for each individual image recording is a major problem, since an overall time for the measurement cannot be extended by the position determination, on account of the bleaching of the sample. Furthermore, it must be possible to use any given positions in any given sequence in order to obtain a particular signal form of the position in the depth direction. Therefore, the basic problem which the present invention proposes to solve is to provide a method wherein image recording of a position of the sample is possible with any given position variation in the depth direction and without any delay.